Phosphoinositides are membrane phospholipids that control many cellular events and bind with variable levels of specificity to dozens of intracellula proteins. Defects in production or metabolism of these anionic phospholipids are associated with cancer, neuronal defects, and other diseases. The most abundant phosphoinositide in most cells types, PI4,5P2, is particularly important for regulation of cytoskeletal assembly during cell motility, differentiation and proliferation largely through its effects on numerous actin binding proteins including gelsolin, N-WASP, and formins, which are the focus of this project. Other isomers of PI4,5P2, such as PI3,5P2, are associated with neurodegeneration in several genetically distinct diseases. How specific phosphoinositides affect their ligands is much less understood than are the mutations that produce abnormal phosphoinositide production. Defining how these lipids exert their biological control at the membrane-cytoskeletal interface could lead to new approaches to limiting or reversing the abnormal function of these lipids in disease. Previous work and preliminary data show that PI4,5P2 at low mole fraction in membranes forms nanoscale clusters in the presence of physiologically relevant levels of Ca2+ and partitions into the liquid disordered phase when membranes undergo fluid phase transitions due to changes in cholesterol content or temperature. Redistribution of PI4,5P2 into these nanodomains alters its ability to nucleate actin assembly from brain extracts and to inhibit gelsolin, the actin filament severing protein. The goal of this project is to quantitatively define the conditions under which PI4,5P2 reorganizes into nanoscale membrane domains using a combination of high resolution imaging, spectroscopy, and molecular dynamics computations and to relate changes in PI4,5P2 membrane distribution to its ability to inhibit actin assembly. Biochemical analyses will test which elements of the actin regulatory system are affected when PI4,5P2 redistributes in membranes. This project involves collaboration among three groups with complementary experience in membrane biophysics, gelsolin biochemistry and the mechanics of actin polymerization; computational studies of membrane structure and mechanics; and electron microscopy with emphasis on high resolution studies of actin assembly at membranes. The multi-disciplinary study will lead to an atomic level understanding of phosphoinositide-protein interactions that will help direct strategies designed to alter phosphoinositide production, distribution, and signaling in the numerous contexts where their altered expression or distribution is linked to disease.